Project Summary/Abstract: Solid tumor metastasis has extremely low long-term survival rate and is currently responsible for over 90% of cancer deaths. Recently, T cells bearing engineered chimeric antigen receptors (CAR-T cells) have showed tremendous promise in treating hematological cancers but still face tremendous challenges in treating solid cancers. In particular , due to low-level systemic expression of most tumor antigens, conventional CAR-T can be activated at non-tumor sites leading to major ?on-target/off-tumor' side- effects (OTOT). In this work we propose to increase CAR-T targeting efficiency and specificity for solid tumors and decrease the OTOT side-effects by activating CAR-T cells in a context-dependent manner. Recent studies have demonstrated that the mechanical properties of cancer microenvironments including stiffness tightly Therefore, we hypothesize that the unique mechanical properties of the metastatic niche offer an intriguing target for the development of mechanosensing CAR-T that specifically target solid tumors and their metastases. By coupling CAR expression (e.g., anti-HER2 CARs for breast cancer) to mechano-sensitive promoters, we expect to trigger T cell activation specifically in the stiffness- correlate to breast cancer metastasis. specific tumor microenvironment. Targeting both cancer antigens AND tumor biophysical microenvironmental cues would significantly reduce the `on target/off tumor' side-effects, and allow for increased T cell dosages and efficacy, collectively leading to improved patient outcomes. This project will involve 1) constructing and characterizing CAR-Ts that specifically respond to stiffness in vitro, and 2) validating whether mechano- sensing CAR-Ts specifically activate in stiff tumor microenvironment in vivo to treat breast cancer lung proposed study could potentially lead to next generation CAR-T therapies that uniquely target metastases. This the mechano-niche of metastases for the treatment of metastatic breast cancer and potentially any types of solid cancer metastasis.